JP2004525106A - Probiotic compounds obtained from Lactobacillus casei strain KE01 - Google Patents

Abstract

Probiotic compositions are provided that comprise Lactobacillus casei strain KE01 having ATCC accession number PTA-3945. The disclosed probiotic compositions are useful in inhibiting enteric pathogen diseases in animals and in maintaining animal health. Methods of making and using Lactobacillus casei strain KE01 probiotic compositions are also disclosed as are methods of using these probiotic compositions.

Description

ＤＮＡ抽出方法
ＷｉｚａｒｄゲノムＤＮＡ精製キット（Promega, WI, USA）を用いてラクトバシラスからＤＮＡを抽出した。簡単に言えば、１ｍＬの２４時間成長させたＭＲＳブロスの各ラクトバシラス種の培養物を遠心分離により回収し、細胞を５０ｍＭのＥＤＴＡ中で再懸濁し、１０ｍｇ／ｍＬリゾチーム（Sigma, MO, USA）で３７℃で６０分間処理した。ラクトバシラス種杆菌細胞を遠心分離でペレット化し、上清を除去した。細菌のペレットを核溶解溶液中で再懸濁し、８０℃で５分間インキュベートした。細胞懸濁液を室温に冷却し、ＲＮアーゼをその溶液に混合した。その懸濁液を３７℃で６０分間インキュベートした。インキュベーション後、タンパク質沈殿溶液をその混合物に加えた。溶液をボルテックスで混合し、氷上で５分間インキュベートした。その混合物を３分間１５Ｋ×ｇで遠心分離し、上清を新しいチューブに移し、ＤＮＡをイソプロピルアルコールで沈殿させた。ＤＮＡを遠心分離し、イソプロピルアルコールを吸引した。ＤＮＡペレットを７０％エタノールで洗浄し、遠心分離で回収した。エタノールを除去し、ペレットを乾燥させた。ＤＮＡをトリス−ＥＤＴＡ緩衝液中で再懸濁した。
【００４４】
抽出されたＤＮＡのＰＣＲ増幅
Cocconcelli等（１９９５）に従い、１マイクロリッターの抽出されたＤＮＡを用いて、単一の任意のヌクレオチド配列を用いてｉＣｙｃｌｅｒ（Bio-Rad, CA, USA）でＰＣＲ反応を行った。２×ＰＣＲ溶液−Ｐｒｅｍｉｘ Ｔａｑ（Takara, Shiga, Japan）を各反応に用いた。各反応は、総量５０μＬ、１．２５ユニットのＴａｋａｒａ Ｅｘ Ｔａｑ ＤＮＡポリメラーゼ、１×緩衝液、２００μＭのｄＮＴＰミックス（それぞれ２．５ｍＭ）を含んでいた。プライマーの最終濃度を４μＭとし、増幅に用いられたプライマーは、５’−ＡｇＣＡｇＣｇＴｇｇ−３’であり(Operon Technologies. Inc., CA, USA)、テンプレートＤＮＡを含む反応混合物を、以下の温度プロフィールでサイクル反応させた：９４℃で５分間を１サイクル；９４℃で１分間、２９℃で１分間、１．５分間で７２℃まで上昇させ、７２℃で１．５分間維持を４０サイクル；７２℃で２分間を１サイクル；および、４℃で維持。[Cocconcelli, PS et al., Development of RAPD protocol for typing of strains of lactic acid bacteria and entercocci. Lett. Appl. Microbiol. 21:376-379 (1995)]。
【００４５】
ゲル電気泳動
各ＲＡＰＤで増幅された反応物のアリコート（１０μＬ）を、Sambrook等（１９８９）に従って、トリス−ホウ酸−ＥＤＴＡ緩衝液中の１％（ｗｔ／ｖｏｌ）のアガロースゲル電気泳動により分析した。ゲルを冷却しないで１２０Ｖで２時間泳動させた。標準としてＤＮＡ分子量マーカーのＨｙｐｅｒｌａｄｄｅｒ Ｉ（Bioline, Randolph, MA, USA）を用いた。電気泳動した後、ゲルをエチジウムブロマイド（５μｇ／ｍＬ）で１０分間染色し、５分間洗浄し、可視化し、Ｆｌｕｏｒ−Ｓ ＭｕｌｔｉＩｍａｇｅｒ（BioRad, CA, USA）で分析した。[Sambrook, J., Fritsch, E.F., Maniatis, T. Molecular Cloning - A Laboratory Manual, 2^nd Edition. Cold Spring Harbor Laboratory Press, New York (1989)]。
【００４６】
単一の１０−ｍｅｒプライマーを用いたＲＡＰＤ分析により、様々なラクトバシラス種参照株およびＫＥ０１株のＤＮＡサンプルを含む１％アガロースゲル上で、変化する強度の、多数の増幅産物のはっきりとした固定パターンが得られた。ゲル上での、異なる種の断片と参照ラクトバシラス種との比較を示した。図１および表２で示された説明文付きの固定パターンは、ＫＥ０１株を一意的に同定し、ゲノムフィンガープリンティングライブラリーを提供するのに役立つ。ゲノムフィンガープリンティングに基づいて、図２で示されたようにデンドグラム（dendogram）を推測した。系統発生分析のワードのクラスター方法を適用した。この方法は、各工程で形成され得る２種のクラスターのいずれかの平方和を最小化し、小さいサイズのダスター（duster）を作製した。系統発生分析に基づき、ＫＥ０１株は、ラクトバチルス−ヘルヴェティクスＡＴＣＣ１５００９と１３％相同性を示し、ラクトバシラス−カゼイ属の亜種ラムノーサスＡＴＣＣ７４６９と５５％相同性を示した。
【００４７】
ラクトバシラス−カゼイＫＥ０１株の純粋な培養物をアメリカンタイプカルチャーコレクション（Bethesda, MD）に寄託し、ＡＴＣＣ番号が付与された。
【００４８】
【表２】

II ．本発明の実施例
以下の実施例で用いられたバルク状ラクトバシラスＫＥ０１株を、発酵微生物学の当業者に既知の標準的な発酵方法を用いて大量生産した。次に、精製したラクトバシラス種ＫＥ０１株を、様々な送達システム、例えば、以下に示すような、微粉末、巨丸剤カプセル、ゲル、および乾燥動物飼料混合物の調製物に用いた。
【００４９】
実施例１
本発明のラクトバシラスＫＥ０１株組成物の調製
凍結乾燥ＫＥ０１の微粉末形態への調製
−２２℃で凍結保存されたラクトバシラス種ＫＥ０１の純粋な培養物（開始物）を、タンパク質、ビタミン、ミネラルおよび炭水化物源を含む発酵ブロス培地中で再生させた。ＮＢＳ１５００Ｌ発酵器に取り付けられた発酵槽で種培養を調製した。発酵プロセス中、微生物の純度を、所定の時間ポイントで（対数期と最終サイクルとを通して）モニターした。微生物の集団を殺菌した分離器で回収し、無水デキストロースおよびトレハロースの混合物を含む担体と混合した後、細胞濃縮物のスラリーを凍結乾燥した。凍結保護物質として、噴霧乾燥したグレードＡの低温加熱した無脂肪低温殺菌乳を用いた。凍結乾燥したＫＥ０１濃縮物を、殺菌した製粉器具を用いて微粉末に粉砕した。使用前に、ＫＥ０１粉末の品質の純度および生存率を確かめた。
【００５０】
ＫＥ０１粉末の混合物への混合：
全ての操作を、温度（７４〜７６°Ｆ）および湿度（３０〜４０％）に制御した環境で行った。ＫＥ０１粉末（約１０^１１ＣＦＵ／ｇのラクトバシラス）、および、下記の組成に列挙された他の成分を、混合するために閉鎖系に導入した。一連のジオメトリック希釈を行い、全ての成分が均質に分配されているかを確認した。水底の分流とクロスフローとを確実にするために、カスタマイズされたジオメトリーでマスフロー容器回転システム（mass flow bin tumbling system）内で混合した。混合したら、５ｌｂの円筒型容器にパッケージングするまでその粉末を閉鎖系で維持した。混合した粉末を、吸湿パックまたは内在の防湿特性を有し、気密蓋で密閉された適切な容器（円筒形容器、単位用量パックなど）に充填する。
【００５１】
「約」という用語は、バルク材料を計量したり製造したりする際に固有のエラーの妥当な要素を与えるのに用いられることは、当業者に明白である。本明細書で用いられるように「約」とは、０．１〜０．５％の誤り要因を含み、さらに、最終組成物を配合するのに用いられた材料の量は、トータルで１００％でなければならず、１００％を超えることはできない、ということは当業者に明白である。全てのパーセントは、重量パーセントを基礎とする。例えば、限定を意図しないが、本発明の一実施形態において、本発明の教示に従って製造されたプロバイオティック組成物は、１グラム当たり約１０^５〜１０^１１ＣＦＵのＫＥ０１を含む約１〜５％のＫＥ０１粉末、および、９５〜９９％の不活性成分または活性成分を含み、当該不活性成分または活性成分は、これらに限定されないが、炭水化物、脂質、ポリペプチド、脂肪酸、植物性化学物質（phytochemical）、および、それらの組み合わせからなる群より選択される。
【００５２】
本発明の教示に従って用いることができる炭水化物としては、単糖類、二糖類、オリゴ糖類、および、多糖類、例えば、これらに限定されないが、トレハロース、マルトース、スクロース、デキストロース、ラクトース、イヌリン、リボース、麦芽デキストリン等が挙げられる。本発明の一実施形態において、二糖類は、トレハロース二水和物であり、オリゴ糖はフルクトオリゴ糖であり、多糖類は麦芽デキストリンである。
【００５３】
適切な脂質としては、これらに限定されないが、大豆油、オリーブ油、パーム核油、落花生油、クルミ油、キャノーラ油（cannola oil；菜種油）等が挙げられる。適切なポリペプチドとしては、ホエータンパク質、卵アルブミン、ゼラチン、乳タンパク質、ならびに、他の動物タンパク質および植物タンパク質が挙げられる。最後に、本明細書で用いられる植物性化学物質としては、ポリフェノール類、サポニン類、フラボノイド類、モノテルペン類、硫化アリル類、リコピン類、カロチノイド類、ポリアセチレン（polyactetylenes）、シリマリン、グリシルリジンカテキン類などのような化合物が挙げられる。
【００５４】
本発明の一実施形態において、ラクトバシラス−カゼイＫＥ０１株プロバイオティックは、以下の重量パーセントの成分を含む。
【００５５】
【表３】

ＫＥ０１の巨丸剤カプセルへの調製
凍結乾燥ＫＥ０１微粉末（約１０^５〜１０^１１ＣＦＵ／ｇのラクトバシラス）、トレハロース二水和物（食品グレード）、および、デキストロース（食品グレード）または麦芽デキストリン（Grain Processing Corp., Muscatine, IA）を、それぞれ３％：６７％：３０％で、徹底的に混合した。混合した混合物をゼラチンカプセルに充填した。使用前に、ＫＥ０１巨丸剤カプセルの品質を純度および生存率に関して試験した。
【００５６】
【表４】

ゲル形態のＫＥ０１の調製
凍結乾燥ＫＥ０１微粉末（約１０^５〜１０^１１ＣＦＵ／ｇのラクトバシラス）を、２．５％濃度でフルクトオリゴ糖（医薬品グレード）と徹底的に混合した。非ＧＭＯ（遺伝子組換え生物）を押出／排除した大豆油を第一の担体としてを用いた。ゲルを徹底的に混合し、滅菌したへらを用いてバッチの上部、中心部および下部からサンプルを回収し、ＫＥ０１の純度および生存率に関して分析した。ＫＥ０１ゲルを、使用するための５０／ｃｃのチューブに充填した。
【００５７】
【表５】

実施例２
細胞付着研究
インビトロでの放射付着分析（ＲＡＡ）を行い、Ｃｅｌｌ Ｅｎｖｉｒｏｎｍｅｎｔｓ（登録商標）（Becton Dickinson, Bedford, Mass.）の、I型コラーゲン(Cn)、IV型Cn、ラミニン、またはフィブロネクチンを含む２４−ウェルプレート、および、Ｃａｃｏ−２細胞単層を用いて、ラクトバシラス種ＫＥ０１株の付着を測定した。
【００５８】
ラクトバシラスＫＥ０１細胞の調製
Ｂａｃｔｏ（登録商標）ラクトバシラスＭＲＳブロス（Difco）中で嫌気性条件下で一晩培養したＫＥ０１の培養物の、５０μｌの接種材料を、１０ｍｌのＭＲＳブロス（^３Ｈ−チミジン（２０μｃｉ）を含む）中に再接種した。ＫＥ０１細胞を、嫌気性条件下で、３７℃で、対数期（約７時間）まで培養し、^３Ｈ−チミジンを細菌ＤＮＡへ最適に摂取させ、取り込まさせた。^３Ｈ−チミジンで標識したＫＥ０１を７，５００×ｇでの遠心分離により回収し、洗浄し、リン酸緩衝食塩水（ＰＢＳ、ｐＨ７．２）中に再懸濁した。細菌細胞密度を、６００ｎｍで光学的に１０^７個のラクトバシラス／ｍｌになるように調節した。
【００５９】
放射付着分析（ＲＡＡ）
上皮下部のマトリックスの相互作用分析のために、Ｃｅｌｌ Ｅｎｖｉｒｏｎｍｅｎｔｓ（登録商標）の、Ｉ型コラーゲン（Ｃｎ）、IV型Ｃｎ、ラミニン、またはフィブロネクチンを含む２４−ウェルプレートを用いて、ＫＥ０１の付着を試験した。マトリックス成分を、平らで光学的に透明なコーティングとして塗布し、このコーティングは総表面積の１．７５ｃｍ^２をカバーした。バイオコート（登録商標）マトリゲルマトリックス、Ｅｎｇｅｌｂｒｅｔｈ−Ｈｏｌｍ−Ｓｗａｒｍマウスの腫瘍から抽出した可溶性基底膜（室温で再生すると、ゲルを形成する）も、相互作用の研究に用いられた。２ミリリットルのＫＥ０１懸濁液（２×１０^７個のラクトバシラス）を、マトリックス層を含む各ウェルに加え、室温でインキュベートした。１時間後、ＫＥ０１懸濁液をウェルから吸引し、廃棄した。１ミリリットルのトリプシン１型（１１０酵素ユニット；Sigma Chemicals, St. Louis, MO.）を、各ウェルに加え、室温で３０分間加水分解させ、マトリックスタンパク質層を解離させた。トリプシン加水分解産物をシンチレーションバイアルに吸引し、各ウェルからの追加の１ｍｌを１０分間室温でホモジナイズした（Ｓｃｉｎｔｉｇｅｓｔ（登録商標）；Fisher Scientific, Fair Lawn, NJ）。ホモジネートを対応するシンチレーションバイアルに吸引した。１０ｍｌの量のシンチレーションカクテル（Ｓｃｉｎｔｉｓａｆｅ（登録商標）Ｇｅｔ、Fisher Scientific）をバイアルに分注し、徹底的に混合した。混合物を沈殿させ、清澄化した後、液体シンチレーション分析器（Ｔｒｉ−Ｃａｒｂ２１００ＴＲ（登録商標）、Packard Inc.）を用いて放射能を測定した。
【００６０】
真核細胞の相互作用分析に関して、Ｃａｃｏ−２、結腸癌腫細胞系を、２４−ウェルの組織培養プレートで、７２時間、ＣＯ２インキュベーターで、イーグル最小必須培地（１％非必須アミノ酸および１０％ウシ胎仔血清が添加された）を用いて単層の集合になるまで培養した。完全な単層が得られた後、各プレートをリン酸緩衝食塩水（ＰＢＳ、ｐＨ７．２）で２回洗浄した。２ｍｌ量（約２×１０^７個の細菌）の^３Ｈ−チミジンで標識したラクトバシラス懸濁液を、約１０^５個のＣａｃｏ−２細胞を含む各ウェルに加えた。Ｃａｃｏ−２細胞とラクトバシラスとの比率を１：２００に維持した。３７℃で１時間インキュベートした後、細菌ｌ懸濁液を吸引し、ウェルをＰＢＳで洗浄した。各ウェルを１型トリプシン、組織ホモジナイザーで処理し、上述したように放射能を測定した。結合をラクトバシラスの結合として示した、生物学的表面の面積１ｃｍ^２あたり（上皮下部のマトリックスタンパク質の相互作用のため）、または、細胞１個あたり（Ｃａｃｏ−２細胞の相互作用のため）。
【００６１】
また、ＫＥ０１と異なるバイオマトリックス層との相互作用を、表６に記載の３つのＡＴＣＣ参照株、ラクトバシラス−カゼイＡＴＣＣ３９３、ラクトバシラス−ロイテリＡＴＣＣ２３２７２、および、ラクトバシラス−ラムノーサスＡＴＣＣ７４６９の相互作用と比較した。有意性を決定するため、分散（variance）の４×６階乗（factorial）分析を用いて結合データを分析した（SAS Inc., Raleigh, NC）。
【００６２】
【表６】

ＫＥ０１は、インビトロで、Ｉ型コラーゲン、IV型コラーゲン、ラミニン、および、フィブロネクチン、および、マトリゲル（登録商標）層に強い結合を示した。Ｃａｃｏ−２細胞単層に結合したＫＥ０１は、約５４％（１０８個のラクトバシラス／細胞）であった。ＫＥ０１と、上皮下部のマトリックス層およびＣａｃｏ−２細胞単層とのインビトロでの相互作用の全ては、ＡＴＣＣ参照株、ラクトバシラス−カゼイＡＴＣＣ３９３（ｐ＜０．０００１）、ラクトバシラス−ロイテリＡＴＣＣ２３２７２（ｐ＜０．０００１）、およびラクトバシラス−ラムノーサスＡＴＣＣ７４６９（ｐ＜０．０００１）より顕著に高かった。
【００６３】
実施例３
腸管病原体の細胞付着の妨害の研究
ＫＥ０１の効能は、腸管病原体（表７に列挙）のバイオマトリックスへの付着を阻害し、病原体−バイオマトリックスの複合体を解離することである。
【００６４】
【表７】

付着−解離の分析
ｔｒｙｐｔｉｃ ｓｏｙ ｂｒｏｔｈ（ＴＳＢ）中で一晩培養した大腸菌Ｏ１５７：Ｈ７の培養物、または、適切な培地培養液中で培養された他の列挙された病原体の、１０μｌの接種材料を、３ｍｌのＴＳＢまたは対応する適切な培地（^３Ｈ−チミジン（２０μｃｉ）を含む）中に再接種した。Ｉ型コラーゲン（Ｃｎ）、IV型Ｃｎ、ラミニン、フィブロネクチンおよびマトリゲル表面を含むバイオコート（登録商標）プレートへの細菌性病原体の結合を、実施例１で説明されたように行った。これらのバイオコート（登録商標）−細菌病原体複合体に、２ｍｌ量の標識されていないＫＥ０１懸濁液（２．０×１０^７個のラクトバシラス／ｍｌ）を各ウェルに対して加え、１時間室温でインキュベートした。ラクトバシラスＫＥ０１懸濁液をウェルから吸引した。以下の工程、すなわちウェル中の内容物の放出の工程、および、放射能測定の工程は、実施例２で説明されたプロトコールに従ってなされた。ウェル中の付着した病原体の細菌細胞数の差（ＫＥ０１処理有り、および、無し）を、「Ｌｏｇ_１０解離」値として示した。
【００６５】
付着−阻害の分析
標識されていないＫＥ０１のバイオコート（登録商標）プレート（Ｉ型コラーゲン（Ｃｎ）、IV型Ｃｎ、ラミニン、フィブロネクチンおよびマトリゲル表面を含む）への結合を実施例２で説明されたように行った。これらのバイオコート（登録商標）−ＫＥ０１複合体に、２ｍｌ量の^３Ｈ−チミジンで標識した大腸菌または他の細菌病原体の懸濁液（２．０×１０^７個の細菌／ｍｌ）を各ウェルに対して加え、１時間室温でインキュベートし、懸濁液を吸引した。以下の工程、すなわちウェル中の内容物の放出の工程、および、放射能測定の工程は、実施例２で説明されたプロトコールに従ってなされた。ＫＥ０１で処理されていないバイオコート（登録商標）ウェルは、大腸菌または他の病原体における付着のコントロールとして提供された。ＫＥ０１で前処理された細菌性病原体のバイオマトリックスへの付着の減少を、「Ｌｏｇ_１０阻害」値として示した。
【００６６】
大腸菌Ｏ１５７：Ｈ７（ＡＴＣＣ４３８９５株）とバイオマトリックスおよびマトリゲル（登録商標）バイオコート表面との相互作用における、ＫＥ０１の阻害および解離効果の結果を表８に示す。
【００６７】
【表８】

ＫＥ０１は、試験された全てのバイオ表面への強い結合を示したが、Ｉ型コラーゲンおよびマトリゲル（登録商標）との相互作用は顕著に高かった。ＫＥ０１は、大腸菌Ｏ１５７：Ｈ７のＩ型コラーゲンおよびマトリゲル（登録商標）へ付着の、＞３−ｌｏｇ阻害を引き起こした；その一方で、IV型コラーゲン、フィブロネクチンおよびラミニン相互作用については＞２−ｌｏｇ阻害を引き起こした。付着した大腸菌のＫＥ０１による解離の効能は、阻害値と同様のＬｏｇ値であった。
【００６８】
結合を阻害し付着した細菌を解離するＫＥ０１の効能の範囲を、表９で列挙された数種の腸管病原体を用いて試験した。
【００６９】
【表９】

ＫＥ０１は、２．８−ｌｏｇ（エンテロコッカス-フェカーリスについて）〜４．２−ｌｏｇ（サルモネラ−エンテリティディスについて）の範囲で、腸管病原体の相互作用を効果的に阻害した。比較してみると、腸管病原体を解離するＫＥ０１の効能は、３．１−ｌｏｇ（腸管出血性大腸菌ＡＴＣＣ４３８８９について）〜４．４−ｌｏｇ（腸管出血性大腸菌ＡＴＣＣ４３８９０について）の範囲で、より高かった。
【００７０】
実施例４
付着した腸管病原体を解離するＫＥ０１微粉末の効能
付着した腸管病原体を解離するＫＥ０１微粉末の効能を、Ｃａｃｏ−２単層を用いた細胞系で試験した。腸管病原体に関するＣａｃｏ−２細胞の付着分析を、実施例２でＫＥ０１について説明したように行った。付着−解離分析を、実施例３でバイオマトリックス層について説明したように行った。しかしながら、上記の２つのプロトコールにおいて、ＫＥ０１粉末を、１％溶液（１００ｍｌの生理的食塩水、ｐＨ７．２中に再懸濁され、徹底的にボルテックスされた１ｇの微粉末）として用いた。生理食塩水での処理をコントロールとして用い、その値を結果を解釈する際にバックグラウンドとして差し引いた。データを表１０に示す。
【００７１】
【表１０】

Ｃａｃｏ−２細胞の腸管病原体の付着プロファイルは、１．１×１０^６（腸管出血性大腸菌ＡＴＣＣ４３８８８を有するＣａｃｏ−２細胞１個あたり、１５個の細菌）〜６．７×１０^６（サルモネラ−プローラムＡＴＣＣ１３０３６を有するＣａｃｏ−２細胞１個あたり、８９個の細菌）の範囲であった。ＫＥ０１は、Ｃａｃｏ−２細胞に付着した腸管病原体複合体を有効に解離し、その解離効能は、１．３−ｌｏｇ（９３．５％）〜３．１−ｌｏｇ（９９．９％）（それぞれ大腸菌ＡＴＣＣ４３８８８およびサルモネラ-プローラムについて）の範囲であった。
【００７２】
実施例５
新鮮なＫＥ０１および凍結乾燥ＫＥ０１調製物がウシの腸管の上皮粘膜に付着および定着する能力の証明
ウシの腸の付着分析：
新しく屠殺した動物から腸サンプルを得て、冷蔵状態で研究所へ移送した。腸を切り開き、内腔の上皮粘膜を穏やかに洗浄し、糞の屑を除去した。以下の２種の異なるＫＥ０１調製物、すなわちＡ）実施例２で説明された^３Ｈ−チミジンで標識したＫＥ０１（約１０^７個のラクトバシラスを含む接種材料の０．１ｍｌ）；および、Ｂ）実施例１で説明されたＫＥ０１粉末ブレンド混合物（生理食塩水で均一に懸濁され、懸濁された、約１０^７個のラクトバシラスを含む接種材料の０．１ｍｌ）を、１インチ^２の粘膜表面に接種した。２時間インキュベートした後、接種した領域を穏やかに生理食塩水で３回洗浄した。
【００７３】
サンプル調製物−ＡをＫＥ０１付着に関して試験した。接種領域を切り出し、組織ホモジナイザーで消化（digested）し、液体シンチレーションフルイドを用いて増幅し、実施例２で説明された放射付着分析に従って放射能を測定した。ＫＥ０１は、ウシの腸上皮粘膜への強い結合、すなわち、約２．５×１０^６個のラクトバシラス／インチ^２を示した。
【００７４】
サンプル調製物−ＢのＫＥ０１定着を、走査型電子顕微鏡法（ＳＥＭ）により評価した。試験片を２％ＯｓＯ_４で３０〜６０分間処理し、水でリンスした。試験片を５０％、７０％および９５％エタノールで各５分間で処理し、続いて２×１０分間１００％エタノールでリンスした。液体二酸化炭素で臨界点乾燥した後、試験片をマウントして、金、パラジウムでスパッタコーティングした。生検試験片をＪＥＯＬ６３００走査型電子顕微鏡を用いて試験した。顕微鏡観察を図３および４に示す。
【００７５】
実施例６
子豚に投与されたＫＥ０１含有動物飼料−サプリメントのインビボでのプロバイオティック効果。ＫＥ０１の腸の定着および糞放出の証明：ブタの糞臭（糞の硫化物およびアンモニア含量の減少）を減少させるＫＥ０１の能力：および、動物の体重増加へのＫＥ０１寄与率
飼料−サプリメント投与量
飼料−サプリメント投与用量を、実施例１で説明したようにゲル形態で調製した。簡単に言えば、凍結乾燥ＫＥ０１微粉末（約１０^５〜１０^１１ＣＦＵ／ｇのラクトバシラス）を、２．５％濃度のフルクトオリゴ糖（ＦＯＳ）と共に徹底的に混合した。非ＧＭＯ（遺伝子組換え生物）を押出／排除した大豆油を第一の担体としてを用いた。ゲルを徹底的に混合し、滅菌したへらを用いてバッチの上部、中心部および下部からサンプルを回収し、ＫＥ０１の純度および生存率に関して分析した。ＫＥ０１ゲルを、使用するための５０／ｃｃのチューブに充填した。また、担体のみ、ＫＥ０１細胞を混合した担体、および、ＫＥ０１細胞とＦＯＳとを混合した担体を含むコントロール飼料投与量を調製した。
【００７６】
動物飼料−サプリメント試験
研究には、４０〜６０ｌｂの範囲の重量のトータルで２０匹のブタ（６〜８週齢）を用いた。これら動物を４つのグループ（それぞれ５匹の動物を含む）に分配し、以下のように分類された。
【００７７】
グループ１（コントロール）：第一の担体のみを含むゲル１０ｃｃを与えた動物。
グループ２：第一の担体と混合した２．５％ＦＯＳのみを含むゲル１０ｃｃを毎日与えた動物。
【００７８】
グループ３：第一の担体と混合した約１０^１０ｃｆｕ／ｃｃのＫＥ０１株細胞のみを含むゲル１０ｃｃを毎日与えた動物。
グループ４：第一の担体と混合した、１０^１０ｃｆｕ／ｃｃのＫＥ０１株細胞と、２．５％ＦＯＳとの組み合わせを含むゲル１０ｃｃを毎日与えた動物。
【００７９】
動物をグループで分け、隔離されたおりに収容し、規則正しく高タンパク質のブタの食餌を与えた。４つの動物グループに、それらそれぞれの分類の飼料−サプリメント投与量（１０ｃｃ）を毎日を与えた。実験を６週間行った。飼料−サプリメント試験中、糞分析および体重測定を毎週規則正しく行った。
【００８０】
糞の回収
糞サンプル（動物当たり約２０ｇ）を滅菌５０ｃｃポリスチレンチューブに回収した。サンプルを正常生理的食塩水で１：１（ｗ／ｖ）の比率で希釈し、徹底的にボルテックスブレンダーでホモジナイズし、２Ｋ×ｇで５分間遠心分離した。上清を慎重にデカントし、微生物学的および化学的分析にかけた。
【００８１】
ベースラインの微生物学的計数（大腸菌型の糞およびラクトバシラス型の糞）および糞中アンモニア含量および糞中硫化物含量のベースラインのレベルを得る実際の飼料−サプリメント試験の１日前に、糞サンプルを回収した。
【００８２】
糞の微生物学的分析
糞溶液（１：１（ｗ／ｖ）のデカントされた上清）を、正常生理的食塩水で１０倍まで連続的に希釈した。Ａｕｔｏｐｌａｔｅ（登録商標）４０００装置（Spiral Biotech, Norwood, MA）を用いて、ＭＲＳ寒天（Difco）上に（全てのラクトバシラス計数のため）、および、ＭａｃＣｏｎｋｅｙ寒天（Difco）上に（全ての大腸菌型計数のため）、選択された希釈物を二連でプレーティングした。Spiral社のオートプレーターを用いて指数対数希釈設定（exponential log dilution setting）でプレーティングを行った。寒天プレートを、それぞれ３７℃（ＭａｃＣｏｎｋｅｙ寒天）および３２℃（ＭＲＳ寒天）で２４〜４８時間インキュベートした。自動化赤外線Ｑ−計数装置（Spiral Biotech, Norwood, MA）を用いて総コロニー計数を概算した。データを糞１グラム当たりの細菌として示し、結果を、ラクトバシラス計数については図５に、大腸菌型の糞の計数については図６に示した。
【００８３】
ＫＥ０１を含む飼料−サプリメント、および、ＫＥ０１とＦＯＳとの組み合わせを含む飼料−サプリメントを投与された動物は、図５で示すように、コントロールグループに比べて、ラクトバシラス種杆菌の糞放出において約２．５−ｌｏｇの増加を示し、ブタ消化管においてＫＥ０１株の増殖および定着を示した。その一方で、大腸菌型の糞の計数は、図６で示すように、コントロール動物と比べて、ＫＥ０１を与えられた動物、および、ＫＥ０１＋ＦＯＳを与えられた動物において、約２．５−ｌｏｇの減少を示し、ブタ腸において、ＫＥ０１株による有効な競合的な排除を示した。
【００８４】
糞の硫化物およびアンモニア分析
糞における硫化物およびアンモニア量を、ＭＰ２３０ ｐＨ／イオンメーターを用いたイオン選択的電極で測定した（Mettler Toledo, Columbus, Ohio）。
【００８５】
硫化物イオン選択的電極の標準化のために、７５ｍｌの希釈硫化物標準（１０ｐｐｍ）溶液を、２５ｍｌの硫化物抗酸化剤緩衝液に穏やかに撹拌しながら加えた。ＤＸ２００参照電極と組み合わせたＤＸ２３２銀選択的電極を用いて、溶液の電極電位（Ｅ１）を測定した。７５ｍｌの硫化物（１００ｐｐｍ）溶液、および、２５ｍｌの硫化物抗酸化剤緩衝液を別々に用いて、Ｅ２を測定した。Ｅ１とＥ２との差により、硫化物電極のスロープが構成された。サンプル測定のために、１００ｍｌの標準（１００ｐｐｍ）銀溶液と、２ｍｌの臭化物ＩＳＡ（イオン強度調節）緩衝液と混合し、Ｅ１値を測定した。１０ｍｌの糞溶液と上記溶液とを混合することによって、Ｅ２値を測定した。Ｅ１とＥ２との差をΔＥとみなした。濃度比率（Ｑ）チャートに従い、方程式：Ｃ＝Ｃ_ｓ×Ｑ（Ｃ＝糞中の硫化物濃度；Ｃ_ｓ＝既知の硫化物濃度、すなわち１００ｐｐｍ標準；および、Ｑ＝濃度比率）を用いて、標準スロープとΔＥとに基づき糞中の硫化物濃度を推定した。
【００８６】
アンモニアイオン選択的電極の標準化のために、１ｍｌのアンモニア標準（１０００ｐｐｍ）溶液を、２ｍｌのアンモニアｐＨ／ＩＳＡ（イオン強度調節）緩衝液を予め混合した１００ｍｌの蒸留水に穏やかに撹拌しながら加えた。アンモニアＤＸ２１７電極を用いて、溶液の電極電位（Ｅ１）を測定した。続いて、その溶液に１０ｍｌのアンモニア（１０００ｐｐｍ）標準を加え、電極電位（Ｅ２）の変化を測定した。Ｅ１とＥ２との差により、アンモニア電極のスロープが構成された。サンプル測定のために、１０ｍｌの糞溶液を、２ｍｌのアンモニアｐＨ／ＩＳＡ緩衝液を予め混合した９０ｍｌの蒸留水に、穏やかに撹拌しながら加え、Ｅ１を測定した。糞溶液にアンモニア標準を加え、Ｅ２を測定した。糞中のアンモニア濃度を上述の方程式を用いて推定した。
【００８７】
ＫＥ０１を含む飼料−サプリメント、および、ＫＥ０１とＦＯＳとを含む飼料−サプリメントを投与された動物は、図７および８に示すように、コントロールグループ動物に比べて、顕著な、約３５ｐｐｍ／ｇｍのアンモニア減少、および、約３７５ｐｐｍ／ｇｍの硫化物減少を示した。また、ＫＥ０１を与えられた動物、および、ＫＥ０１とＦＯＳとを与えられた動物からの糞は、増殖したプロバイオティック性ラクトバシラスＫＥ０１による乳酸生成のために、酸臭（rancid）がする。
【００８８】
動物体重増加測定
体重増加に関するベースライン測定を得るために実際に飼料−サプリメント試験を行う１日前に、動物の体重を測定した。全ての４つのグループからの動物の体重を、６週間の飼料試験を通して毎週１回測定した。実験の結果、図９で示したように、動物のコントロールグループに比べて、ＫＥ０１を与えられた動物は約２８ｌｂの体重増加を示し、ＫＥ０１とＦＯＳとを与えられた動物グループは２０ｌｂの体重増加を示した。
【００８９】
III ．結果
最後に、本明細書で開示された本発明の実施形態は本発明の原理を説明していることが理解される。用いられ得る他の改変は、本発明の範囲に含まれる。従って、一例として、これらに限定されないが、本発明の教示に従って、本発明のその他の形状を用いてもよい。従って、本発明は、厳密に示されたもの、説明されたものに限定されない。
【００９０】
本発明を説明する文章中（特に以下の請求項の文章中）で用いられた用語「a」および「an」および「the（該）」ならびに類似の指示語は、明細書中に特に他の規定がない限り、または、文章により明確に否定されない限り、単数および複数の両方を含むと解釈される。本明細書における値の範囲の記述は、その範囲内に含まれるそれぞれ別々の値を個別に意味する簡略的な方法として提供されることを単に意図する。明細書中に特に他の規定がない限り、それぞれの個々の値は、本明細書で個々に列挙されたかのように、本明細書に含まれる。本明細書に記載の全ての方法は、明細書中に特に他の規定がない限り、あるいは、文章により明確に否定されない限り、適切な順番のいずれかで行うことができる。本明細書で示されるありとあらゆる実施例または典型的な言葉（例えば「such as」）の使用は、単に、本発明をより良く説明することを意図しており、本発明の範囲あるいは特許請求された範囲に限定を与えない。本明細書における言葉は、本発明の実施に必須な、特許請求されていない要素のいずれかを示すものと解釈されるべきではない。
【００９１】
本明細書で開示された本発明の代替の要素または実施形態の分類は、限定とは解釈されない。各群の要素は、個々に、または、本明細書で見出される群または他の要素の他の要素との組み合わせのいずれかにおいて、言及され、特許請求され得る。便宜上および／または特許性の理由で、群の１またはそれ以上の要素が含まれてもよいし、または、群から削除されてもよいと予想される。このような包含または削除のいずれかがなされる場合、明細書は、ここに改変された群を含むものとみなされ、従って、添付の請求項で用いられた全てのマーカッシュ群の記載された説明を満たす。
【００９２】
本発明の好ましい実施形態が本明細書に記載されており、本発明者等が知る本発明を実施する最良の形態を含む。もちろん、その好ましい実施形態の改良型は、上述の説明を読めば当業者に明白である。本発明者は、当業者がこのような改良型を適切なものとして用いることを考慮しており、本発明者等は、本発明が特に本明細書に記載されたものとは異なって実施されることを意図する。従って、本発明は、準拠法により認可された本明細書に添付された請求項に記載の主題の全ての改変および同等物を含む。その上、全ての可能なそれらの改変型における上述した要素の組み合わせのいずれかは、明細書中に特に他の規定がない限り、あるいは、文章により明確に否定されない限り、本発明に含まれる。
【００９３】
その上、本明細書を通して、特許および印刷された出版物に対して多数の参照がなされている。上記で引用された参考文献および印刷された出版物はそれぞれ、参照により個々に本明細書に含まれる。
【図面の簡単な説明】
【００９４】
【図１】図１は、ランダム増幅多型ＤＮＡ（ＲＡＰＤ）分析に基づいて、１２種の異なるラクトバシラス種タイプの株と比較した１％アガロースゲル上のラクトバシラス−カゼイＫＥ０１株のゲノムフィンガープリントを示す。
【図２】図２は、ゲノムフィンガープリンティングから推測された系統発生学的系統樹と、ラクトバシラス種ＫＥ０１株とラクトバシラス種タイプの株の他の種との関連を示す。
【図３】図３は、ウシの腸上皮へのラクトバシラス−カゼイＫＥ０１株の付着を示す。
【図４】図４は、ウシの腸上皮粘膜に生体フィルムを形成した定着を示す。
【図５】図５は、本発明の教示に従って動物の食餌にラクトバシラス−カゼイＫＥ０１株を添加した結果として、強化されたラクトバシラスの腸での定着および糞放出をグラフで示す。
【図６】図６は、本発明の教示に従って動物の食餌にラクトバシラス−カゼイＫＥ０１株を添加した結果の、大腸菌型の糞の計数の減少をグラフで示す。
【図７】本発明の教示に従ってに動物の食餌にラクトバシラス−カゼイＫＥ０１株を添加した結果としての、動物の糞におけるアンモニア生成物の減少をグラフで示す。
【図８】図８は、本発明の教示に従って動物の食餌をラクトバシラス−カゼイＫＥ０１株に添加した結果としての、動物の糞における硫化物生成物の減少をグラフで示す。
【図９】図９は、本発明の教示に従って動物の食餌にラクトバシラス−カゼイＫＥ０１株を添加した結果としての、動物における体重増加の上昇をグラフで示す。[0001]
Related application
This application claims priority to US Provisional Application No. 60/256528, filed December 18, 2000, the entire contents of which are incorporated herein by reference.
【Technical field】
[0002]
The present invention discloses a probiotic Lactobacillus casei preparation. In particular, an anti-infective and anti-diarrheal preparation obtained from a newly characterized Lactobacillus casei strain designated KE01 is disclosed. Also disclosed are related methods for preparing probiotic compounds using Lactobacillus casei strain KE01 and related methods for using KE01 probiotic compositions.
[Background Art]
[0003]
The newly recognized probiotic Lactobacillus casei strain described herein has been designated KE01. Previously, this same organism was designated Lactobacillus casei KE99 (see, for example, US Provisional Application No. 60/256528 and AS Naidu, X. Xie, DA Leumer, S. Harrison, MJ Burrill and EA Fonda). 2001. Reduction of sulfide, Ammonia Compounds and Adhesion Properties of Lactobacillus casei strain KE99 In Vitoro. Curr. Microbiol. 43: In press).
[0004]
Lactic acid bacteria (LAB) are a unique microflora in the mammalian gastrointestinal tract that plays an important role in host microecology, and are trusted with an impressive list of therapeutic and prophylactic properties. These therapeutic and prophylactic properties include, but are not limited to, maintaining gut microbial ecology, physiological effects, immunomodulatory effects and antibacterial effects. Other LAB-related properties include enzyme release into the intestinal lumen, which can act synergistically with LAB attachment and alleviate the symptoms of intestinal malabsorption. In addition, LAB enzymes can help regulate intestinal pH, thereby increasing aromatic amino acid degradation. [Fuller, R. Probioticfoods-current use and future developments.IFI NR 3: 23-26 (1993); Mitsuoka, T. Taxonomy and ecology of Bifidobacteria.Bifidobacteria Microflora 3:11 (1984); Gibson, GR et al., Probiotics and intestinal infections, p.10-39.In R. Fuller (ed.), Probiotics 2: Applications and practical aspects.Chapman and Hall, London, UK (1997); Naidu AS, et al., Probiotic spectra of lactic acid bacteria (LAB). Crit Rev Food Sci Nutr 39: 3-126 (1999); Naidu, AS, Clemens, RA Probiotics. p.431-462. In AS Naidu (ed.), Natural Food Antimicrobial Systems. CRC Press , Boca Raton, FL (2000)].
[0005]
Lactic acid bacteria have also demonstrated the ability to reduce odor and toxicity from animal excrement by significantly reducing sulfide and ammonia containing compounds in animal feces waste. This ex vivo use of LAB is becoming more and more important as the agricultural business develops and the community sees endless entry into unused agricultural areas. For example, if rooster and shellfish waste is mixed with a mixture containing 15% carbohydrates and LAB, LAB removes unpleasant odors and reduces hydrogen sulfide production on hatchery waste. It has been shown. Moreover, LAB compositions have been shown to be effective in reducing E. coli and Salmonella content in hatchery waste to negligible levels. In addition, lactic acid fermentation mediated by L. acidophilus significantly reduces the smell and viscosity of poultry waste, such as broiler waste. In addition, LAB-containing preparations have been reported to promote the destruction of hard-to-degrade carbohydrates and to reduce ammonia production by pig caecal bacteria. Finally, fermentation of stored grasses of Lactobacillus casei FG1 and L. plantarum FG10 ex vivo significantly reduces ammonia levels by suppressing ureolytic organisms. [Deshmukh, AC, Patterson, PH Preservation of hatchery wastes by lactic acid fermentation. 1. Laboratory scale fermentation.Poult Sci 76: 1212-1219 (1997); Russell, SM et al., Lactic acid fermentation of broiler processing waste: physical properties and chemical analyzes.Poult Sci 71: 765-770 (1992); Tibbetts, GW et al., Poultry offal ensiled with Lactobacillus acidophilus for growing and finishing swine diets.J Anim Sci 64: 182-190 (1987); Sakata, T. et al., Probiotic preperations dose-dependentty increase net production rates of organic acids and decrease that of annmonia by pig cecal bacteria in batch culture.Dig Dis Sci 44: 1485-1493 (1999); Cai, Y. et al. , Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics, aerobic deterioration of silage.J Dairy Sci 82: 520-526 (1999); Modler, HW et al., Bifidobacteria and bifidogenic factors.Can Inst Food Sci Tech 23 : 29-41 (1990)].
[0006]
However, the greatest potential of LAB to improve the quality of life of humans and domestic animals lies in the in vivo probiotic use of LAB. In order for LAB to exhibit beneficial in vivo probiotic effects, organisms must survive long periods in the gastrointestinal tract. Therefore, it is important to select a probiotic LAB strain that can retain an amount that will prevent rapid clearance by bowel contraction. An effective probiotic bacterium must be able to survive in the gastric environment and be capable of at least temporarily colonizing the gut by attaching to the intestinal epithelium. As a result, LABs exhibiting enhanced ability to adhere to mucosal surfaces, and thus exhibiting improved bacterial retention and sustained gastrointestinal residence time, are more likely than LABs that do not. And has competitive advantages. [Salminen, S. et al., Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges.Antonie Van Leeuwenhoek 70: 347-358 (1996); Conway, P. Selection criteria for probiotic microorganisms. Asia Pacific J Clin Nutr 5: 10-14 (1996); Havenaar, R. et al., Selection of strains for probiotic use, p.209-224.In R. Fuller (ed.), Probiotics, the scientific basis.Chapman and Hall, London, UK (1992)].
[0007]
Lactobacillus species can successfully colonize the mammalian digestive tract via a number of different mechanisms. For example, some bacterial species bind to various sub-epithelial matrix proteins and specific receptors on intestinal mucosa. Other species can attach to mammalian intestinal cells on the eukaryotic cell surface of bacteria and hosts via mechanisms that require different combinations of carbohydrates and protein factors. However, regardless of the mechanism of attachment, LAB's ability to successfully colonize the human gastrointestinal tract confers on LAB probiotic properties. [Greene.JD.Klaenhammer, TR Factors involved in adherence of lactobacilli to human Caco-2 cell.Appl Environ Microbiol 60: 4487-4494 (1994); Sarem, F. et al., Comparison of the adherence of three Lactobacillus strains to Caco-2 and Int-407 human intestinal cell lines.Let Appl Microbiol 22: 439-442 (1996); Naidu, AS, et al., Particle agglutination assays for rapid detection of fibronectin, fibriogen, and collagen receptors on Staphylococcus aureus. J Clin Microbiol 26: 1549-1554 (1988); Wadstrom, T. et al., Surface properties of lactobacilli isolated from the small intestines of pigs.J Appl Bacteriol 62: 513-520 (1987); Bernet, MF et al. , Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment, invasion by entero-virulent bacteria.Gut 35: 483-489 (1994); Jin, LZ et al., Effect of adherent Lactobacillus spp. On in vitro adherence of salmonellae to the intestinal epithelial cells of chichen.J Appl Bacteriol 81: 201-206 (1996); Re id, G. et al., Influence of lactobacilli on the adhesion of Staphylococcus aureus and Candida albicans to fibers and epithelial cells, J Ind Microbiol 15: 248-253 (1995)].
[0008]
Generally speaking, probiotic bacteria exert their beneficial effects by displacing invasive or toxin-producing pathogenic enteric bacteria (enteropathogens) from the intestinal mucosa in a competitive binding process. Enteric pathogens, such as, but not limited to, enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), Salmonella enteritidis, Yernia psudotuberculosis, and Listeria monocytogenes must be able to successfully colonize the intestinal tract of animals in order to cause disease.
[0009]
The mechanisms used by these organisms to effectively colonize the gut vary. For example, ETEC with CFA / I or CFA / II adhesin, in particular, adhere to the brush border of Caco-2 cells of dividing human intestinal epithelium in culture. Salmonella typhimurium and EPEC adhere to the brush border of Caco-2 cells in differentiated human intestinal epithelium in culture, whereas Ersina-pseudotuberculosis and S. monocytogenes are undifferentiated. Binds to the periphery of Caco-2 cells.
[0010]
Recently, heat-killed Lactobacillus acidophilus preparations have proven to be effective probiotic compositions. Heat-killed Lactobacillus acidophilus preparations have been shown, depending on dose, to displace known enteric pathogens from the lining of the intestinal wall of test animals. As a result, enteric pathogens could no longer colonize the animal's digestive tract, preventing disease. For example, it has been found that Lactobacillus acidophilus (Lacteol® strain) inhibits the attachment of this E. coli B41 strain (ECB41) in a dose-dependent manner. In other experiments, live Lactobacillus acidophilus LB strain and heat-killed Lactobacillus acidophilus LB strain successfully inhibited both Caco-2 cell association and invasion of enteric pathogens. In still other studies, heat-killed Lactobacillus acidophilus LB strains were shown to completely inhibit ETEC attachment to Caco-2 cells. [Fourniat, J. et al., Heat-kilted Lactobacillus acidophilus inhibits adhesion of Escherichia coli B41 to HeLa cells.Ann Rech Vet 23: 361-370 (1992); Chauviere, G. et al., Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus.FEMS Microbiol Lett 70: 213-217 (1992)]. Coconnier, MH et al., Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2. cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol Lett 110: 299-305 (1993)].
[0011]
As mentioned above, a probiotic composition is generally defined as a microbial dietary supplement that beneficially affects the host by improving the intestinal microbial balance. The two major genera of microorganisms generally associated with probiotics include Lactobacillus species and Bifidobacteria sp. Beneficial effects of probiotics include increased resistance to infections, a healthier immune system, reduced irritable bowel syndrome, reduced blood pressure, reduced serum cholesterol, allergy relief, and tumor regression . However, despite these recent advances in science and limited published in vivo and in vitro experimental evidence supporting the efficacy of probiotic compositions, these and other benefits are reported. The main research has relied on detoxification evidence.
[0012]
Examples of publications and limited in vitro data that rely on detoxification include US Pat. Nos. 3,957,974; 4,210,672; 4,314,995; Nos. 4,345,032; 4,579,734; 4,871,539; 4,879,238 and 5,292,362 ("Hata" patent). Hata's patent discloses various sub-species of Lactobacillus species and reports beneficial probiotic-like effects. However, the Hata patent does not disclose or describe Lactobacillus species that exhibit greedy binding to the subepithelial matrix and competitive elimination, and interference with bacterial enteric pathogens by microorganisms.
[0013]
US Patent No. 6,060,050 ("the '050 patent") discloses a preparation consisting of a probiotic microorganism suspended in a starch-containing medium, including a medium used to protect organisms during storage. Disclosed are useful for transferring organisms to the large intestine and provide a growth promoting substrate. However, the '050 patent does not provide data directly indicating probiotic activity against enteric pathogens.
[0014]
U.S. Pat. No. 5,965,128 ("the '128 patent") discloses the treatment of intestinal diseases caused by E. coli O157: H7 (enterohemorrhagic E. coli). However, the probiotic composition of the '128 patent contains only the non-pathogenic strain E. coli, and no Lactobacillus species is disclosed. As a result, the non-pathogenic intestinal organisms of the '128 patent can provide recipients with "probiotic-like" protection against enterohemorrhagic Escherichia coli, while relating to Lactobacillus species and the genus Bifidobacteria. Lack of other useful features.
[0015]
U.S. Pat. Nos. 4,839,281, 5,032,399 and 5,709,857 (the '857 patent) disclose several Lactobacillus acidophilus strains that adhere to intestinal mucosal cells. . Furthermore, the '857 patent discloses a Lactobacillus acidophilus strain that inhibits the growth of certain enteric pathogens. However, the '857 patent does not disclose a Lactobacillus casei strain that has proven beneficial features with respect to the' 857 patent Lactobacillus acidophilus culture.
[0016]
Therefore, there remains a need for new strains of Lactobacillus species that exhibit probiotic activity. In particular, there is a need for more Lactobacillus strains that have a proven ability to reduce disease caused by enteric pathogens and enhance animal vitality and health.
DISCLOSURE OF THE INVENTION
[Summary of the invention]
[0017]
It is an object of the present invention to provide new strains of Lactobacillus species that have demonstrated probiotic properties.
[0018]
It is another object of the present invention to provide a new strain of Lactobacillus sp. Having probiotic activity of an anti-enteric pathogen.
Yet another object of the present invention is to provide a new strain of Lactobacillus that maintains animal health and vitality.
[0019]
Yet another object of the present invention is to provide dietary supplements and pharmaceutical formulations made from Lactobacillus species having demonstrated probiotic properties.
The present invention achieves these and other objects by providing a new strain of Lactobacillus casei designated KE01, which provides a novel strain of science, such as demonstrated in vivo anti-enteric pathogen activity. It has proven probiotic properties. In addition, the present invention provides a dietary supplement and a pharmaceutical composition comprising Lactobacillus casei strain KE01, which dietary supplement and the pharmaceutical composition provide long-term protection for the organism and its proven In combination with a saccharide complex consisting of trehalose and fructooligosaccharides to help maintain probiotic properties.
[0020]
There is a need for new probiotic formulations that can be used to treat and prevent enteric pathogen infections and help maintain human and livestock health and vitality. In recent years, the U.S. Food and Drug Administration (FDA) has increased its campaign against over-prescription and clinical abuse of antibiotics. Excessive antibiotic use increases with the number of human and animal pathogens that are resistant to first-line antibiotics, thereby increasing infections that do not respond to conventional antimicrobial therapy. In addition, the use of prophylactic antibiotics in animal feeds increases surprisingly the intestinal infection of livestock, and malabsorption of nutrients can reduce herd size and animal weight. As a result, the number of healthy animals suitable for human consumption is reduced, and animals that have survived long enough to market have noticeably lost weight and consequently meat quality .
[0021]
One means of preventing the rapid spread of drug-resistant enteric pathogens in humans and livestock is to significantly reduce the use of antibiotics. However, due to the overcrowding of breeding grounds and cities, the spread of infectious diseases such as intestinal infections is inevitable. As a result, appropriate antimicrobial alternatives must be available before prophylactic antibiotic use is completely discontinued. Recent studies have shown that the use of food and dietary supplements containing certain strains of probiotic microorganisms can help prevent, and often cure, enteropathogenic diseases. It is shown. However, many probiotic formulations currently marketed rely on organisms, including Lactobacillus species and Bifidobacterium species (and other genera), which provide approved methods for assessing probiotic efficacy. The scientific research used has not been performed. As a result, too many of the currently available “probiotic” formulations have not demonstrated anti-intestinal pathogen activity in vivo. In addition, many of the commercially available clinically effective probiotic formulations are not suitable for storage and, therefore, do not deliver an effective amount of viable probiotic bacteria to the user. We have tested a number of commercial preparations and found that the viability of the microorganisms is much lower than the indicated concentration, and in many cases we have found that these commercial It has been found that it does not contain any bacteria.
[0022]
The present inventors have developed a method for preparing and packaging a new strain of Lactobacillus casei designated KE01. The inventor originally obtained this new strain of Lactobacillus casei from a traditional fermented yogurt-like Asian dairy product. We then characterized the isolate and deposited the strain with the American Type Culture Collection (ATCC, MD, USA). Lactobacillus casei strain KE01 was given ATCC accession number PTA-3945. In addition, the inventors have developed a preparation that maintains the viability of Lactobacillus casei KE01 such that a clinically effective amount of viable probiotic microorganism reaches the host.
[0023]
The present invention provides a Lactobacillus casei strain (KE01) that inhibits bacterial adhesion of enteric pathogens (interference with microorganisms), including, but not limited to, a variety of mammalian cell types. Enteropathogenic and enterotoxigenic E. coli, Helicobacter pylori, Campylobacter jejuni, Salmonella typhimurium, and Salmonella enteritidis. Moreover, the lactobacilli of the present invention also prevent these bacterial pathogens and other bacterial pathogens from competitively binding to many mammalian cells (competitive elimination). The beneficial properties of the novel Lactobacillus strains of the present invention have resulted in improved probiotic dietary supplements that support overall human and animal health. In addition, the present invention relates to prophylactic, therapeutic and palliative agents for conditions such as, but not limited to, traveler diarrhea, gastrointestinal infections, hemolytic uremic syndrome, and gastric ulcers (as described herein). In this document, these are collectively referred to as "probiotics."
[0024]
Additional new features and characteristics of this novel Lactobacillus casei strain KE01 include, but are not limited to, exceeding 48 ppm of sulfide concentrations in excess of 300 ppm when exposed to growth media containing about 2000 ppm sulfide. And the ability of Lactobacillus casei KE01 to decrease by a factor that increases the number of subepithelial matrices such as Biocoat® (collagen type I, collagen type IV, laminin, and fibronectin), Matrigel® and Showing strong binding to the monolayer of Caco-2 cells and the like. Most importantly, it was shown that the reconstituted lyophilized preparation of Lactobacillus casei of the present invention effectively detaches E. coli attached to collagen.
[0025]
The methods used to maintain the viability of the Lactobacillus casei of the present invention and retain its E. coli replacement properties include, but are not limited to, the use of the sugar trehalose and water, and the use of an oxygen-tolerant final composition. Packaging in a foil pouch lined with a polymer (eg, Mylar®).
[0026]
These and other beneficial probiotic properties of the new strain of Lactobacillus species are further demonstrated by the following non-limiting detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention comprises about 10 per gram.⁵-10¹¹Dietary supplement containing colony forming units viable Lactobacillus. In another embodiment of the present invention, the dietary supplement comprises about 10 per gram.⁵-10¹¹Containing non-viable Lactobacillus. The Lactobacillus species of the present invention is the Lactobacillus casei strain KE01 having the American Type Culture Collection (ATCC) accession number PTA-3945. The colony forming unit (CFU) of Lactobacillus casei KE01 is equivalent to one bacterial cell. Also, the term colony forming unit or CFU is generally used only when describing a viable bacterium, but when describing an embodiment of the present invention comprised of a non-viable lactobacillus composition, one term is used. Non-viable bacterial cells are also defined.
[0027]
In one embodiment of the invention, the dietary supplement comprises a gelatin capsule filled with a dried Lactobacillus species composition. In another embodiment, the dietary supplement is in the form of a liquid preparation. In yet another embodiment of the present invention, the dietary supplement is in the form of an anal or vaginal suppository. When used as a suppository, the dietary supplement can be formed into affordable boluses, which can include non-toxic lubricants, stabilizers, waxes, and the like, to facilitate administration. In other embodiments of the present invention, the Lactobacillus sp. Dietary supplement can be mixed with food, such as, but not limited to, dairy products, cereals, bread, meat, fruits, vegetables, rice, and the like. The form envisioned by the Lactobacillus dietary supplement of the present invention is not critical and is non-limiting.
[0028]
Throughout this specification, the present invention may be referred to as a probiotic composition, a Lactobacillus seed-containing composition, a dietary supplement, a lyophilized powder, or a Lactobacillus seed composition. All of these above terms are described herein as a viable or non-viable Lactobacillus casei strain KE01 under ATCC accession number PTA-3945, regardless of the presence or absence of form or other components, or herein. A composition comprising genetically equivalents determined using the method is meant.
[0029]
In one embodiment of the invention, the Lactobacillus composition is lyophilized using standard methods known to those skilled in the food sciences. In another embodiment of the Lactobacillus composition of the invention, the Lactobacillus composition is air dried. In yet another embodiment, the Lactobacillus composition is a paste. In still other embodiments, the Lactobacillus compositions of the invention are liquid.
[0030]
The Lactobacillus composition of the present invention may or may not be mixed with additional components. For example, in one embodiment of the present invention, the Lactobacillus composition is mixed with a carbohydrate selected from the group consisting of trehalose, glucose, sucrose, fructose, maltose, and combinations thereof. The carbohydrate may comprise from about 50% to 98% of the total composition. In other embodiments, proteins, such as albumin and / or whey, may or may not be added to the Lactobacillus compositions of the invention.
[0031]
The lactobacillus bacillus composition of the present invention can be taken orally as a bolus in the form of a gelatin capsule, compressed tablet, or gel cup. In another embodiment, the Lactobacillus composition of the present invention can be taken orally in the form of a liquid beverage. Liquid beverages can include other ingredients, such as, but not limited to, flavor enhancers, sweeteners, viscosity enhancers, and other food additives. Further, the present invention can be taken together with other foods, separately or in combination with them.
[0032]
In one embodiment of the invention, the animal is treated with a single dose of the probiotic composition (about 10 per gram).⁵-10¹¹Lactobacilli). Total intake depends on the individual needs of the animal, as well as the weight and size of the animal. Preferred dosages for any given application can be readily determined by titration. The titration is performed using a series of standard weight doses (each about 10⁵-10¹¹Lactobacilli). A series of doses are administered, starting at 0.5 grams and continuing up to the theoretical endpoint determined by the size and dosage form of the animal. Appropriate dosages are achieved when a minimal amount of the Lactobacillus composition that produces the desired result is administered. Appropriate dosages are also known to those skilled in the art as "effective amounts" of the probiotic compositions of the present invention.
[0033]
For example, if it is desired to reduce the symptoms associated with irritable bowel syndrome in an animal, one of the above measured doses may be escalated in successive increments of 0.5 grams each day until the symptoms subside. While daily. In one embodiment of the invention, the preferred dose is about 10 mg / kg body weight (weight of the animal recipient) per day.³-10⁸Viable Lactobacillus range. This is equivalent to about 10 billion viable Lactobacillus casei strains KE01 per day for the average healthy adult. It is straightforward to calculate the approximate dose appropriate for all animals of all weights by extrapolation. It is recognized that this is a non-limiting example that can be suitably varied by those skilled in the art of formulating probiotic compositions by the titration methods described above.
[0034]
The probiotic composition of the present invention can be administered to any animal in need thereof, including, but not limited to, mammals, birds, reptiles, and fish. Typical uses include probiotic compositions of the present invention with humans, horses, pigs, cows, sheep, dogs, cats, rabbits, chickens, turkeys, pheasants, quails, parakeets, parrots, and other wildlife. Administration to animals and livestock animals.
[0035]
In particular, the probiotic compositions of the present invention can be used to inhibit or treat enteropathogen-related diseases when administered to an animal in need thereof using the methods described herein. Diseases caused by enteric pathogens include diseases caused by pathogens such as diarrhea, irritable bowel syndrome and intestinal bleeding. Examples of enteric pathogens associated with these diseases include, but are not limited to, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Salmonella enteritidis, Elsina-Pseudotuberculosis, and Listeria mono. Cytogenes. It is theorized by the inventors that the inhibition and treatment of disease by enteric pathogens through a competitive binding process is achieved by the probiotic compositions of the present invention, but is not provided as a limitation. . That is, the probiotic lactobacilli of the present invention compete with enteric pathogens at binding sites on the intestinal mucosa. Because the probiotic Lactobacillus of the invention has a higher affinity and binding activity for these binding sites than the enteric pathogen, the probiotic Lactobacillus of the invention is harmlessly effluxed from the intestine by normal metabolic processes. And replace the intestinal pathogen with enteric pathogens. In vivo examples of this aforementioned competitive binding and its efficacy in inhibiting and treating diseases by enteric pathogens are provided in the detailed examples below and the accompanying figures.
[0036]
The following technical considerations provide detailed techniques. In section I, one skilled in the art will learn how to isolate and identify probiotic Lactobacillus, in particular, the Lactobacillus casei strain KE01, a probiotic Lactobacillus species of the present invention. In Section II, tests performed to scientifically confirm how to prepare the probiotic compositions of the invention and the probiotic features exhibited by the probiotic compositions of the invention A specific example is provided that teaches It is understood that these specific examples are not intended to be limiting.
[0037]
I. Isolation and characterization of Lactobacillus casei strain KE01
A. Isolation of candidate probiotic bacteria
The probiotic organisms of the present invention were isolated from traditional fermented yogurt-like Asian dairy products. Screening methods have been limited to traditional fermented yogurt-like Asian dairy products with a history of at least 10 years of safe human consumption. Isolation of probiotic bacteria was performed using three selective microbial media using methods known to those skilled in microbiology. Lactobacillus selective medium includes SL medium supplemented with 0.05% cysteine, Bifidobacterium is selected using trypticase phyton yeast extract medium containing antibiotics, and Streptococcus is It was isolated using case yeast extract cysteine medium.
[0038]
Candidate probiotic Lactobacillus strains are catalase-negative, glucose homofermentative, gram-positive, non-spore-forming rods that exhibited low pH, gastric acid and bile tolerance. In addition to the ability of Lactobacillus isolates to grow in media containing acetate, the inability of Lactobacillus isolates to grow at pH 9.0 makes themCarnobacterium speciesHelped to distinguish from. A total of 81 isolates were classified as candidate probiotic lactobacilli based on these criteria and further subdivided into the following criteria: i) low pancreatic juice resistance; ii) subepithelial matrices such as bio Ability to adhere to Coat® Matrigel (Becton Dickinson, Bedford, Mass.) And to cultured intestinal epithelial cells (Caco-2 cell line); iii) Enterohemorrhagic E. coli serum adhering to collagen matrix Type O157: characterized by their ability to competitively eliminate H7; and iv) their ability to reduce ammonia and sulfide containing compounds.
[0039]
After analysis of all 81 candidate probiotic lactobacilli, two strains with all the above mentioned characteristics were identified. These strains were designated as KE97 and KE99 (renamed KE01). Finally, growth and growth rates (generation times determined by impedance detection using a BioMerieux® bactometer system), strain stability in continuous culture, lyophilization and regeneration characteristics, and aroma / flavor characteristics were determined for each. Confirmed by strain.
[0040]
The isolated Lactobacillus casei strain KE01 organism is maintained in a substantially pure culture for use in preparing a probiotic composition of the present invention. As used herein, a "substantially pure culture" refers to a bacteriological culture that produces only one discernable organism when cultured on a solid or semi-solid microbial medium. Means things. Cells of other bacterial species may be present in extremely low amounts; however, these cells are either non-viable, non-cultivable, or subject to typical non-genomic-based microbiological techniques. It will be understood that either below or below the limit of detection used. The term "non-genome-based" is intended to exclude methods such as PCR detection or similar methods used to detect DNA or RNA in microorganisms.
[0041]
Moreover, any powder, paste, gel, bolus, or other probiotic composition of Lactobacillus casei strain KE01 (or simply referred to as KE01) may be produced in accordance with the teachings of the present invention. Is described herein, and it is understood that the Lactobacillus casei strain KE01 was obtained from a substantially pure culture.
[0042]
B. DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis
In the RAPD protocol, PCR is used to create a unique fingerprint for bacterial identification. Analysis by PCR can be performed in a quick and reliable manner. Therefore, RAPD analysis was used for molecular identification and fingerprinting of strain KE01. All 12 Lactobacillus species-type strains from the ATCC collection were fingerprinted and compared to KE01. All Lactobacillus strains were cultured overnight in MRS broth (Difco) for DNA fingerprinting. The Lactobacillus strains analyzed for DNA fingerprints are listed in Table 1.
[0043]
[Table 1]

DNA extraction method
DNA was extracted from Lactobacillus using the Wizard Genomic DNA Purification Kit (Promega, WI, USA). Briefly, cultures of each Lactobacillus species of 1 mL of 24-hour grown MRS broth were harvested by centrifugation, cells were resuspended in 50 mM EDTA, and 10 mg / mL lysozyme (Sigma, MO, USA) At 37 ° C. for 60 minutes. Lactobacillus bacillus cells were pelleted by centrifugation and the supernatant was removed. The bacterial pellet was resuspended in the lysis solution and incubated at 80 ° C. for 5 minutes. The cell suspension was cooled to room temperature and RNase was mixed into the solution. The suspension was incubated at 37 ° C. for 60 minutes. After incubation, a protein precipitation solution was added to the mixture. The solution was vortex mixed and incubated on ice for 5 minutes. The mixture was centrifuged at 15K × g for 3 minutes, the supernatant was transferred to a new tube and the DNA was precipitated with isopropyl alcohol. The DNA was centrifuged and isopropyl alcohol was aspirated. The DNA pellet was washed with 70% ethanol and collected by centrifugation. The ethanol was removed and the pellet was dried. DNA was resuspended in Tris-EDTA buffer.
[0044]
PCR amplification of extracted DNA
According to Cocconcelli et al. (1995), 1 microliter of the extracted DNA was used to perform a PCR reaction on an iCycler (Bio-Rad, CA, USA) using a single arbitrary nucleotide sequence. 2 × PCR solution—Premix Taq (Takara, Shiga, Japan) was used for each reaction. Each reaction contained a total volume of 50 μL, 1.25 units of Takara Ex Taq DNA polymerase, 1 × buffer, 200 μM dNTP mix (2.5 mM each). The final concentration of the primer was 4 μM, the primer used for amplification was 5′-AgCAgCgTgg-3 ′ (Operon Technologies. Inc., CA, USA), and the reaction mixture containing template DNA was subjected to the following temperature profile. Cycling reaction: 1 cycle at 94 ° C. for 5 minutes; 40 cycles of 94 ° C. for 1 minute, 29 ° C. for 1 minute, 1.5 minutes to 72 ° C .; One cycle at 2 ° C for 2 minutes; and maintained at 4 ° C. [Cocconcelli, PS et al., Development of RAPD protocol for typing of strains of lactic acid bacteria and entercocci. Lett. Appl. Microbiol. 21: 376-379 (1995)].
[0045]
Gel electrophoresis
Aliquots (10 μL) of the reactions amplified with each RAPD were analyzed by 1% (wt / vol) agarose gel electrophoresis in Tris-borate-EDTA buffer according to Sambrook et al. (1989). The gel was run at 120 V for 2 hours without cooling. The DNA molecular weight marker Hyperladder I (Bioline, Randolph, MA, USA) was used as a standard. After electrophoresis, the gel was stained with ethidium bromide (5 μg / mL) for 10 minutes, washed for 5 minutes, visualized, and analyzed on a Fluor-S Multilmager (BioRad, CA, USA). [Sambrook, J., Fritsch, E.F., Maniatis, T. Molecular Cloning-A Laboratory Manual, 2^nd Edition. Cold Spring Harbor Laboratory Press, New York (1989)].
[0046]
RAPD analysis using a single 10-mer primer revealed a distinct pattern of immobilization of multiple amplification products of varying intensity on a 1% agarose gel containing DNA samples of various Lactobacillus species reference strains and strain KE01. was gotten. A comparison of fragments of different species with a reference Lactobacillus species on a gel is shown. The descriptive fixed pattern shown in FIG. 1 and Table 2 uniquely identifies the KE01 strain and serves to provide a genomic fingerprinting library. Based on genomic fingerprinting, a dendogram was estimated as shown in FIG. The word cluster method of phylogenetic analysis was applied. This method minimized the sum of the squares of either of the two clusters that could be formed at each step, creating a small size duster. Based on phylogenetic analysis, strain KE01 showed 13% homology with Lactobacillus-Helveticus ATCC 15009 and 55% homology with Lactobacillus casei subsp. Rhamnosus ATCC 7469.
[0047]
A pure culture of Lactobacillus casei strain KE01 was deposited with the American Type Culture Collection (Bethesda, MD) and given an ATCC number.
[0048]
[Table 2]

II . Embodiment of the present invention
The bulk Lactobacillus strain KE01 used in the following examples was mass-produced using standard fermentation methods known to those skilled in fermentation microbiology. The purified Lactobacillus sp. Strain KE01 was then used in various delivery systems, for example, the preparation of fine powders, bolus capsules, gels, and dry animal feed mixtures as shown below.
[0049]
Example 1
Preparation of Lactobacillus strain KE01 of the present invention
Preparation of lyophilized KE01 into fine powder form
A pure culture of Lactobacillus sp. KE01 cryopreserved at −22 ° C. (starting material) was regenerated in a fermentation broth medium containing protein, vitamin, mineral and carbohydrate sources. Seed cultures were prepared in fermenters attached to an NBS 1500 L fermentor. During the fermentation process, the purity of the microorganism was monitored at predetermined time points (through the log phase and the final cycle). The microbial population was collected in a sterile separator, mixed with a carrier containing a mixture of anhydrous dextrose and trehalose, and then the cell concentrate slurry was lyophilized. Spray-dried grade A low-temperature heated non-fat pasteurized milk was used as the cryoprotectant. The lyophilized KE01 concentrate was ground to a fine powder using sterilized milling equipment. Prior to use, the purity and viability of the KE01 powder were checked.
[0050]
Mixing KE01 powder into the mixture:
All operations were performed in a temperature (74-76 ° F) and humidity (30-40%) controlled environment. KE01 powder (about 10¹¹CFU / g of Lactobacillus) and other components listed in the composition below were introduced into the closed system for mixing. A series of geometric dilutions was performed to ensure that all components were homogeneously distributed. Mixing was performed in a mass flow bin tumbling system with customized geometries to ensure bottom diversion and cross flow. Once mixed, the powder was kept in a closed system until packaged in a 5 lb cylindrical container. The mixed powder is filled into a suitable container (cylindrical container, unit dose pack, etc.) having a moisture-absorbing pack or an inherent moisture-proof property and sealed with an airtight lid.
[0051]
It will be apparent to those skilled in the art that the term "about" is used to provide a reasonable component of the inherent error in weighing and manufacturing bulk material. As used herein, “about” includes from 0.1 to 0.5% error factor, and furthermore, the amount of material used to formulate the final composition is 100% total. It should be clear to those skilled in the art that they must be and cannot exceed 100%. All percentages are on a weight percent basis. For example, but not by way of limitation, in one embodiment of the present invention, a probiotic composition made in accordance with the teachings of the present invention comprises about 10 to about 10 grams per gram.⁵-10¹¹About 1-5% KE01 powder, including CFU KE01, and 95-99% inert or active ingredients, including, but not limited to, carbohydrates, lipids, poly It is selected from the group consisting of peptides, fatty acids, phytochemicals, and combinations thereof.
[0052]
Carbohydrates that can be used in accordance with the teachings of the present invention include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including, but not limited to, trehalose, maltose, sucrose, dextrose, lactose, inulin, ribose, malt Dextrin and the like. In one embodiment of the invention, the disaccharide is trehalose dihydrate, the oligosaccharide is fructooligosaccharide, and the polysaccharide is malt dextrin.
[0053]
Suitable lipids include, but are not limited to, soybean oil, olive oil, palm kernel oil, peanut oil, walnut oil, cannola oil (rapeseed oil), and the like. Suitable polypeptides include whey protein, egg albumin, gelatin, milk protein, as well as other animal and plant proteins. Finally, the phytochemicals used in this specification include polyphenols, saponins, flavonoids, monoterpenes, allyl sulfides, lycopenes, carotenoids, polyacetylenes, silymarin, glycyrrhizin catechin Such as compounds.
[0054]
In one embodiment of the present invention, the Lactobacillus casei strain KE01 probiotic comprises the following weight percentage components:
[0055]
[Table 3]

Preparation of KE01 into pill capsule
Lyophilized KE01 fine powder (about 10⁵-10¹¹CFU / g Lactobacillus), trehalose dihydrate (food grade), and dextrose (food grade) or malt dextrin (Grain Processing Corp., Muscatine, IA) at 3%: 67%: 30%, respectively. Mix thoroughly. The mixed mixture was filled into gelatin capsules. Prior to use, the quality of KE01 bolus capsules was tested for purity and viability.
[0056]
[Table 4]

Preparation of KE01 in gel form
Lyophilized KE01 fine powder (about 10⁵-10¹¹CFU / g of Lactobacillus) was thoroughly mixed with fructooligosaccharide (pharmaceutical grade) at a 2.5% concentration. Non-GMO (genetically modified organism) extruded / excluded soybean oil was used as the first carrier. The gel was mixed thoroughly and samples were collected from the top, center and bottom of the batch using a sterile spatula and analyzed for KE01 purity and viability. KE01 gel was filled into 50 / cc tubes for use.
[0057]
[Table 5]

Example 2
Cell adhesion research
In vitro radioadhesion analysis (RAA) was performed and a 24-well plate containing type I collagen (Cn), type IV Cn, laminin, or fibronectin from Cell Environments® (Becton Dickinson, Bedford, Mass.). And the adhesion of Lactobacillus sp. Strain KE01 was measured using a Caco-2 cell monolayer.
[0058]
Preparation of Lactobacillus KE01 cells
A 50 μl inoculum of a culture of KE01 grown overnight under anaerobic conditions in Bacto® Lactobacillus MRS broth (Difco) was added to 10 ml of MRS broth (³H-thymidine (containing 20 μci). KE01 cells are cultured under anaerobic conditions at 37 ° C. until log phase (about 7 hours),³H-thymidine was optimally taken up and incorporated into bacterial DNA.³KE01 labeled with H-thymidine was collected by centrifugation at 7,500 xg, washed and resuspended in phosphate buffered saline (PBS, pH 7.2). Bacterial cell density is determined optically at 600 nm by 10⁷Lactobacilli / ml.
[0059]
Radiation Attachment Analysis (RAA)
Test KE01 attachment using 24-hour plates containing Cell Environments® type I collagen (Cn), type IV Cn, laminin, or fibronectin for subepithelial matrix interaction analysis did. The matrix component is applied as a flat, optically clear coating that has a total surface area of 1.75 cm²Covered. Biocoat® Matrigel matrix, a soluble basement membrane extracted from tumors of Engelbreth-Holm-Swarm mice (which forms a gel when regenerated at room temperature) was also used for interaction studies. 2 ml of KE01 suspension (2 × 10⁷Lactobacilli) were added to each well containing the matrix layer and incubated at room temperature. After 1 hour, the KE01 suspension was aspirated from the wells and discarded. One milliliter of trypsin type 1 (110 enzyme units; Sigma Chemicals, St. Louis, MO.) Was added to each well and hydrolyzed at room temperature for 30 minutes to dissociate the matrix protein layer. The trypsin hydrolyzate was aspirated into scintillation vials and an additional 1 ml from each well was homogenized for 10 minutes at room temperature (Scintigest®; Fisher Scientific, Fair Lawn, NJ). The homogenate was aspirated into the corresponding scintillation vial. Scintillation cocktail (Scintisafe® Get, Fisher Scientific) in a volume of 10 ml was dispensed into vials and mixed thoroughly. After allowing the mixture to settle and clarify, the radioactivity was measured using a liquid scintillation analyzer (Tri-Carb2100TR®, Packard Inc.).
[0060]
For eukaryotic cell interaction analysis, Caco-2, a colon carcinoma cell line, was cultured in 24-well tissue culture plates for 72 hours in a CO2 incubator in Eagle's minimal essential medium (1% non-essential amino acids and 10% fetal calf). (Serum was added) to form a monolayer assembly. After a complete monolayer was obtained, each plate was washed twice with phosphate buffered saline (PBS, pH 7.2). 2ml volume (about 2 × 10⁷Bacteria)³H-thymidine-labeled Lactobacillus suspension is added to about 10⁵Was added to each well containing Caco-2 cells. The ratio of Caco-2 cells to Lactobacillus was maintained at 1: 200. After incubation at 37 ° C. for 1 hour, the bacterial suspension was aspirated and the wells were washed with PBS. Each well was treated with type 1 trypsin, tissue homogenizer and radioactivity was measured as described above. 1 cm area of biological surface, indicating binding as lactobacillus binding²Per (for the interaction of matrix proteins below the epithelium) or per cell (for the interaction of Caco-2 cells).
[0061]
In addition, the interaction of KE01 with a different biomatrix layer was compared with the interactions of the three ATCC reference strains shown in Table 6, Lactobacillus casei ATCC 393, Lactobacillus reuteri ATCC 23272, and Lactobacillus rhamnosus ATCC 7469. To determine significance, the binding data was analyzed using a 4 × 6 factorial analysis of variance (SAS Inc., Raleigh, NC).
[0062]
[Table 6]

KE01 showed strong binding to type I collagen, type IV collagen, laminin and fibronectin and Matrigel® layers in vitro. KE01 bound to Caco-2 cell monolayer was about 54% (108 lactobacilli / cell). All of the in vitro interactions of KE01 with the subepithelial matrix layer and the Caco-2 cell monolayer were determined by the ATCC reference strain, Lactobacillus casei ATCC 393 (p <0.0001), Lactobacillus reuteri ATCC23272 (p <0). .0001), and Lactobacillus-Rhamnosus ATCC 7469 (p <0.0001).
[0063]
Example 3
Study of interference with enteric pathogen cell adhesion
The efficacy of KE01 is to inhibit the attachment of enteric pathogens (listed in Table 7) to the biomatrix and dissociate the pathogen-biomatrix complex.
[0064]
[Table 7]

Attach-dissociate analysis
A 10 μl inoculum of a culture of Escherichia coli O157: H7 grown overnight in a tryptic soy broth (TSB), or other listed pathogens grown in a suitable culture medium, was added to 3 ml of TSB or Corresponding appropriate medium (³H-thymidine (containing 20 μci). Bacterial pathogen binding to Biocoat® plates containing type I collagen (Cn), type IV Cn, laminin, fibronectin and Matrigel surfaces was performed as described in Example 1. These Biocoat®-bacterial pathogen complexes were added to a 2 ml volume of unlabeled KE01 suspension (2.0 × 10⁷(Lactobacillus / ml) was added to each well and incubated for 1 hour at room temperature. The Lactobacillus KE01 suspension was aspirated from the wells. The following steps, ie, the step of releasing the contents in the wells, and the step of measuring radioactivity were performed according to the protocol described in Example 2. The difference in the number of bacterial cells of the adhering pathogen in the wells (with and without KE01 treatment) was determined by “Log”.₁₀Dissociation "values.
[0065]
Adhesion-inhibition analysis
Binding of unlabeled KE01 to Biocoat® plates (including type I collagen (Cn), type IV Cn, laminin, fibronectin and Matrigel surface) was performed as described in Example 2. These Biocoat®-KE01 complexes were added in 2 ml volumes.³Suspension of E. coli or other bacterial pathogen labeled with H-thymidine (2.0 x 10⁷Bacteria / ml) was added to each well, incubated for 1 hour at room temperature, and the suspension was aspirated. The following steps, ie, the step of releasing the contents in the wells, and the step of measuring radioactivity were performed according to the protocol described in Example 2. Biocoat® wells that were not treated with KE01 served as a control for attachment in E. coli or other pathogens. The reduction in the attachment of bacterial pathogens pretreated with KE01 to the biomatrix was described in "Log₁₀Inhibition "values.
[0066]
Table 8 shows the results of the inhibition and dissociation effects of KE01 on the interaction between E. coli O157: H7 (ATCC 43895 strain) and the surface of the biomatrix and Matrigel (registered trademark) Biocoat.
[0067]
[Table 8]

KE01 showed strong binding to all biosurfaces tested, but interaction with type I collagen and Matrigel® was significantly higher. KE01 caused> 3-log inhibition of the attachment of E. coli O157: H7 to type I collagen and Matrigel®, while> 2-log inhibition for type IV collagen, fibronectin and laminin interactions. Caused. The efficacy of dissociation of the adhered Escherichia coli with KE01 was a Log value similar to the inhibition value.
[0068]
The range of potency of KE01 to inhibit binding and dissociate attached bacteria was tested using several enteric pathogens listed in Table 9.
[0069]
[Table 9]

KE01 effectively inhibited intestinal pathogen interactions in the range of 2.8-log (for Enterococcus faecalis) to 4.2-log (for Salmonella-entitydis). By comparison, the efficacy of KE01 to dissociate enteropathogens was higher, ranging from 3.1-log (for enterohemorrhagic E. coli ATCC 43889) to 4.4-log (for enterohemorrhagic E. coli ATCC 43890). .
[0070]
Example 4
Efficacy of KE01 fine powder for dissociating attached enteric pathogens
The efficacy of KE01 micropowder in dissociating attached enteric pathogens was tested in a cell line using Caco-2 monolayer. Caco-2 cell adhesion analysis for enteric pathogens was performed as described for KE01 in Example 2. Attachment-dissociation analysis was performed as described for the biomatrix layer in Example 3. However, in the above two protocols, KE01 powder was used as a 1% solution (1 g of fine powder resuspended in 100 ml saline, pH 7.2 and thoroughly vortexed). Saline treatment was used as a control and the value was subtracted as background when interpreting the results. The data is shown in Table 10.
[0071]
[Table 10]

The Caco-2 cell intestinal pathogen adhesion profile was 1.1 × 10⁶(15 bacteria per Caco-2 cell with enterohemorrhagic E. coli ATCC 43888)-6.7 x 10⁶(89 bacteria per Caco-2 cell with Salmonella-prolam ATCC13036). KE01 effectively dissociates the enteric pathogen complex attached to Caco-2 cells, and its dissociation efficacy is from 1.3-log (93.5%) to 3.1-log (99.9%) (each E. coli ATCC 43888 and Salmonella-proram).
[0072]
Example 5
Demonstration of the ability of fresh and freeze-dried KE01 preparations to adhere and colonize bovine intestinal epithelial mucosa
Bovine intestinal adhesion analysis:
Intestinal samples were obtained from newly sacrificed animals and transported refrigerated to the laboratory. The intestine was cut open and the luminal epithelial mucosa was gently washed to remove fecal debris. The following two different KE01 preparations: A) described in Example 2³KE01 labeled with H-thymidine (about 10⁷B) KE01 powder blend mixture as described in Example 1 (uniformly suspended in saline, about 10%).⁷0.1 ml of the inoculum containing 1 lactobacillus)²Was inoculated on the mucosal surface. After a two hour incubation, the inoculated area was gently washed three times with saline.
[0073]
Sample preparation-A was tested for KE01 adhesion. The inoculated area was excised, digested with a tissue homogenizer, amplified using liquid scintillation fluid, and measured for radioactivity according to the radioadhesion analysis described in Example 2. KE01 has strong binding to bovine intestinal epithelial mucosa, ie, about 2.5 × 10⁶Lactobacilli per inch²showed that.
[0074]
KE01 fixation of Sample Preparation-B was evaluated by scanning electron microscopy (SEM). Specimen is 2% OsO₄For 30-60 minutes and rinsed with water. Specimens were treated with 50%, 70% and 95% ethanol for 5 minutes each, followed by a 2x10 minute rinse with 100% ethanol. After drying at the critical point with liquid carbon dioxide, the test piece was mounted and sputter coated with gold and palladium. Biopsy specimens were examined using a JEOL 6300 scanning electron microscope. Microscopic observations are shown in FIGS.
[0075]
Example 6
In vivo probiotic effects of KE01-containing animal feed-supplements administered to piglets. Evidence of KE01 intestinal colonization and fecal release: ability of KE01 to reduce pig fecal odor (reduction of fecal sulfide and ammonia content): and the contribution of KE01 to animal weight gain
Feed-supplement dosage
Feed-supplement doses were prepared in gel form as described in Example 1. Briefly, lyophilized KE01 fine powder (about 10⁵-10¹¹CFU / g of Lactobacillus) was thoroughly mixed with 2.5% strength fructooligosaccharide (FOS). Non-GMO (genetically modified organism) extruded / excluded soybean oil was used as the first carrier. The gel was mixed thoroughly and samples were collected from the top, center and bottom of the batch using a sterile spatula and analyzed for KE01 purity and viability. KE01 gel was filled into 50 / cc tubes for use. In addition, a control feed dose containing only the carrier, a carrier in which KE01 cells were mixed, and a carrier in which KE01 cells and FOS were mixed were prepared.
[0076]
Animal feed-supplement testing
The study used a total of 20 pigs (6-8 weeks old) weighing in the range of 40-60 lb. The animals were divided into four groups, each containing five animals, and were classified as follows.
[0077]
Group 1 (control): animals receiving 10 cc of gel containing only the first carrier.
Group 2: Animals receiving daily 10 cc of a gel containing only 2.5% FOS mixed with the first carrier.
[0078]
Group 3: about 10 mixed with the first carrier¹⁰Animals fed daily with 10 cc of gel containing only cfu / cc KE01 cells.
Group 4: 10 mixed with the first carrier¹⁰Animals fed daily with 10 cc of a gel containing a combination of cfu / cc KE01 cell line and 2.5% FOS.
[0079]
Animals were divided into groups, housed in isolated cages, and regularly fed a high protein pig diet. Four groups of animals received their respective class of feed-supplement doses (10 cc) daily. The experiment was performed for 6 weeks. During the diet-supplement test, fecal analysis and body weight measurements were performed regularly weekly.
[0080]
Collection of feces
Fecal samples (about 20 g per animal) were collected in sterile 50 cc polystyrene tubes. Samples were diluted with normal saline at a ratio of 1: 1 (w / v), thoroughly homogenized in a vortex blender, and centrifuged at 2K × g for 5 minutes. The supernatant was carefully decanted and subjected to microbiological and chemical analysis.
[0081]
One day prior to the actual feed-supplementation test to obtain baseline microbiological counts (E. coli-type feces and Lactobacillus-type feces) and baseline levels of fecal ammonia and fecal sulfide content, fecal samples were collected Collected.
[0082]
Microbiological analysis of feces
The fecal solution (1: 1 (w / v) decanted supernatant) was serially diluted up to 10-fold with normal saline. Using an Autoplate® 4000 instrument (Spiral Biotech, Norwood, Mass.) On MRS agar (Difco) (for all Lactobacillus counts) and on MacConkey agar (Difco) (all E. coli type counts). Selected dilutions were plated in duplicate. The plating was performed using an exponential log dilution setting using an autoplater from Spiral. The agar plates were incubated at 37 ° C. (MacConkey agar) and 32 ° C. (MRS agar) for 24-48 hours, respectively. Total colony counts were estimated using an automated infrared Q-counter (Spiral Biotech, Norwood, MA). The data are shown as bacteria per gram of feces, and the results are shown in FIG. 5 for Lactobacillus counts and in FIG. 6 for E. coli type fecal counts.
[0083]
As shown in FIG. 5, animals receiving the diet-supplement containing KE01 and the diet-supplement containing the combination of KE01 and FOS had about 2. It showed a 5-log increase, indicating growth and colonization of strain KE01 in the porcine gastrointestinal tract. On the other hand, the count of E. coli-type feces was reduced by about 2.5-log in the animals given KE01 and the animals given KE01 + FOS, as shown in FIG. And showed effective competitive elimination by the KE01 strain in pig intestine.
[0084]
Fecal sulfide and ammonia analysis
Sulfide and ammonia levels in feces were measured with an ion selective electrode using a MP230 pH / ion meter (Mettler Toledo, Columbus, Ohio).
[0085]
For standardization of sulfide ion selective electrodes, 75 ml of diluted sulfide standard (10 ppm) solution was added to 25 ml of sulfide antioxidant buffer with gentle stirring. The electrode potential (E1) of the solution was measured using a DX232 silver selective electrode in combination with a DX200 reference electrode. E2 was measured using 75 ml of sulfide (100 ppm) solution and 25 ml of sulfide antioxidant buffer separately. The difference between E1 and E2 constituted the slope of the sulfide electrode. For sample measurement, 100 ml of standard (100 ppm) silver solution was mixed with 2 ml of bromide ISA (ion intensity control) buffer and the E1 value was measured. The E2 value was measured by mixing 10 ml of the fecal solution with the above solution. The difference between E1 and E2 was considered as ΔE. According to the concentration ratio (Q) chart, the equation: C = C_s× Q (C = sulfide concentration in feces; C_s= Known sulfide concentration, ie 100 ppm standard; and Q = concentration ratio), the sulfide concentration in feces was estimated based on the standard slope and ΔE.
[0086]
For standardization of the ammonia ion selective electrode, 1 ml of ammonia standard (1000 ppm) solution was added with gentle stirring to 100 ml of distilled water premixed with 2 ml of ammonia pH / ISA (Ionic Strength Adjustment) buffer. . The electrode potential (E1) of the solution was measured using an ammonia DX217 electrode. Subsequently, 10 ml of ammonia (1000 ppm) standard was added to the solution, and the change in electrode potential (E2) was measured. The difference between E1 and E2 constituted the slope of the ammonia electrode. For sample measurement, 10 ml of fecal solution was added to 90 ml of distilled water premixed with 2 ml of ammonia pH / ISA buffer with gentle stirring and E1 was measured. An ammonia standard was added to the fecal solution, and E2 was measured. The ammonia concentration in feces was estimated using the above equation.
[0087]
The animals receiving the feed-supplement containing KE01 and the feed-supplement containing KE01 and FOS showed a marked increase in ammonia concentration of about 35 ppm / gm compared to the control group animals, as shown in FIGS. And a sulfide reduction of about 375 ppm / gm. In addition, feces from animals receiving KE01 and animals receiving KE01 and FOS have a rancid odor due to the production of lactic acid by the probiotic lactobacillus KE01 grown.
[0088]
Animal weight gain measurement
The animals were weighed one day before the actual feed-supplementation test to obtain a baseline measurement for weight gain. Animals from all four groups were weighed weekly throughout a 6-week diet test. As a result of the experiment, as shown in FIG. 9, the animals receiving KE01 gained about 28 lb of weight compared to the control group of animals, and the animal group receiving KE01 and FOS gained about 20 lb of weight. showed that.
[0089]
III . result
Finally, it is understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that can be used are within the scope of the invention. Thus, by way of example, and not limitation, other shapes of the invention may be used in accordance with the teachings of the invention. Accordingly, the invention is not limited to what has been strictly shown and described.
[0090]
The terms "a" and "an" and "the" as well as similar designations used in the text describing the invention, particularly in the text of the following claims, particularly refer to other words in the specification. Unless otherwise provided, or unless otherwise expressly negated by the text, it is to be construed to include both the singular and the plural. The description of a range of values herein is merely intended to be provided as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise stated in the specification, each individual value is included herein as if individually listed herein. All methods described herein can be performed in any suitable order, unless otherwise specified in the specification, or unless the context clearly dictates otherwise. The use of any and all examples or exemplary terms (eg, "such as") provided herein is merely intended to better describe the invention and is not intended to limit the scope of the invention or the claims. It does not limit the scope. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0091]
Classifications of the alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed, either individually or in combination with other members of the group or other members found herein. It is anticipated that one or more members of the group may be included or deleted from the group for convenience and / or patentability reasons. In the event that any such inclusion or deletion is made, the specification will be deemed to include the modified group herein and, thus, the written description of all Markush groups used in the appended claims. Meet.
[0092]
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, modifications of that preferred embodiment will be apparent to those of skill in the art upon reading the above description. The inventor considers that those skilled in the art will use such modifications as appropriate, and they believe that the present invention may be practiced otherwise than as specifically described herein. Intended to be. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated in the specification or expressly negated by the text.
[0093]
Moreover, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications is individually incorporated herein by reference.
[Brief description of the drawings]
[0094]
FIG. 1 shows the genomic fingerprint of Lactobacillus casei KE01 strain on a 1% agarose gel compared to 12 different Lactobacillus species type strains based on random amplified polymorphic DNA (RAPD) analysis. .
FIG. 2 shows the phylogenetic phylogenetic tree inferred from genomic fingerprinting and the association of Lactobacillus sp. Strain KE01 with other species of Lactobacillus sp. Type strains.
FIG. 3 shows the attachment of Lactobacillus casei strain KE01 to bovine intestinal epithelium.
FIG. 4 shows colonization of a bovine intestinal epithelial mucosa with a biological film.
FIG. 5 graphically illustrates enhanced intestinal colonization and fecal release of Lactobacillus casei strain KE01 as a result of adding the Lactobacillus casei strain KE01 to animal diets in accordance with the teachings of the present invention.
FIG. 6 graphically illustrates the reduction in E. coli-type fecal counts as a result of adding Lactobacillus casei strain KE01 to animal diets in accordance with the teachings of the present invention.
FIG. 7 graphically illustrates the reduction of ammonia products in animal feces as a result of adding Lactobacillus casei strain KE01 to animal diets in accordance with the teachings of the present invention.
FIG. 8 graphically illustrates the reduction of sulfide products in animal feces as a result of adding animal diet to Lactobacillus casei strain KE01 according to the teachings of the present invention.
FIG. 9 graphically illustrates the increase in weight gain in animals as a result of adding Lactobacillus casei strain KE01 to the animal's diet in accordance with the teachings of the present invention.

Claims (33)

A probiotic composition comprising a Lactobacillus casei strain KE01 having ATCC accession number PTA-3945, wherein the Lactobacillus casei strain KE01 is derived from a substantially pure culture. object. 2. The probiotic composition of claim 1, further comprising an inert or active ingredient selected from the group consisting of carbohydrates, polypeptides, lipids, phytochemicals, and combinations thereof. 3. The probiotic composition of claim 2, wherein said carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof. 4. The probiotic composition of claim 3, wherein the carbohydrate is selected from the group consisting of trehalose, maltose, sucrose, dextrose, lactose, inulin, ribose, and combinations thereof. 4. The probiotic composition according to claim 3, wherein said disaccharide is trehalose dihydrate. The probiotic composition according to claim 3, wherein the oligosaccharide is a fructooligosaccharide. The probiotic composition according to claim 3, wherein the polysaccharide is malt dextrin. 3. The probiotic composition of claim 2, wherein the polypeptide is selected from the group consisting of whey protein, egg albumin, gelatin, milk protein, and combinations thereof. The probiotic composition according to claim 2, wherein the lipid is selected from the group consisting of soybean oil, olive oil, palm kernel oil, peanut oil, walnut oil, rapeseed oil, and combinations thereof. The phytochemicals are selected from the group consisting of polyphenols, saponins, flavonoids, monoterpenes, allyl sulfides, lycopenes, carotenoids, polyacetylene, silymarin, glycyrrhizinate catechins, and combinations thereof. 3. The probiotic composition according to claim 2, wherein the composition is: The probiotic composition of claim 1, further comprising trehalose. The probiotic composition according to claim 11, further comprising malt dextrin. The probiotic composition of claim 11, further comprising a fructooligosaccharide. 14. The probiotic composition of claim 13, wherein the Lactobacillus casei strain KE01 is present in an amount of about 10 < ⁵ > to 10 < ¹¹ > colony forming units (CFU) per gram. 15. The probiotic composition according to any one of claims 1 to 14, wherein the probiotic composition is a bolus, gel or liquid administered to an animal. 16. The probiotic composition according to claim 15, wherein said animal is selected from the group consisting of mammals, fish, birds and reptiles. 17. The probiotic composition of claim 16, wherein said mammal is selected from the group consisting of humans, horses, dogs, cats, rabbits, sheep, pigs, and cows. 17. The probiotic composition of claim 16, wherein said birds are selected from the group consisting of chickens, turkeys, pheasants, quails, parakeets and parrots. The probiotic composition according to claim 15, wherein the bolus is selected from the group consisting of a gelatin capsule, a compressed tablet, and a gel capsule. 20. The probiotic of claim 19, wherein the bolus is packaged in a polymer-lined foil membrane packet. A powdered Lactobacillus casei strain KE01 having ATCC accession number PTA-3945 in an amount of about 1 to 5 weight percent;
A disaccharide in an amount of about 25-95 weight percent;
About 0 to 10 weight percent oligosaccharide; and
About 0 to 50 weight percent polysaccharide,
A probiotic composition comprising: 22. The probiotic of claim 21, wherein the powdered Lactobacillus casei strain KE01 having ATCC accession number PTA-3945 has about 10 < ⁵ > to 10 < ¹¹ > CFU per gram and is present in an amount of about 3 weight percent. Tick composition. 22. The probiotic composition of claim 21, wherein the disaccharide is present in an amount of about 62 weight percent. 22. The probiotic composition of claim 21, wherein the oligosaccharide is present in an amount of about 5 weight percent. 22. The probiotic composition of claim 21, wherein the polysaccharide is present in an amount of about 30 weight percent. 22. The probiotic composition according to claim 21, wherein said disaccharide is trehalose, said oligosaccharide is fructooligosaccharide, and said polysaccharide is malt dextrin. With about ¹⁰ 5 ^{to 10} 11 CFU and per gram having ATCC Accession No. PTA-3945, about 3 weight percent of powdered Lactobacillus - casei strain KE01;
About 62 weight percent trehalose;
About 5 weight percent fructooligosaccharides; and
About 30 weight percent malt dextrin;
A probiotic composition comprising: 27. Controlling a disease caused by an enteric pathogen in an animal, comprising administering to an animal in need thereof an effective amount of a probiotic composition according to any one of claims 1, 11, 12, 13, 20 or 26. how to. 29. The enteropathogen is selected from the group consisting of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), Salmonella enteritidis, Ersina-pseudotuberculosis and Listeria monocytogenes. The method described in. 29. The method of claim 28, wherein said animal is selected from the group consisting of mammals, fish, birds, and reptiles. 31. The method of claim 30, wherein said mammal is selected from the group consisting of humans, horses, dogs, cats, rabbits, sheep, pigs, and cows. 31. The method of claim 30, wherein the birds are selected from the group consisting of chickens, turkeys, pheasants, quails, parakeets and parrots. 29. The method of claim 28, wherein said administering further comprises a probiotic composition selected from the group consisting of gelatin capsules, compressed tablets, gel capsules, animal feed and liquid beverages.

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